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PubMed2005

Use of Forster's resonance energy transfer microscopy to study lipid rafts.

Rafts in cell membranes have been a subject of much debate and many models have been proposed for their existence and functional significance. Recent studies using Forster's resonance energy transfer (FRET) microscopy have provided one of the first glimpses into the organization of putative raft components in living cell membranes. Here we discuss how and why FRET microscopy provides an appropriate non-invasive methodology to examine organization of raft components in cell membranes; a combination of homo and hetero-FRET microscopy in conjunction with detailed theoretical analyses are necessary for characterizing structures at nanometre scales. Implications of the physical characteristics of the organization of GPI-anchored proteins in cell membranes suggest new models of lipid-based assemblies in cell membranes based on active principles.

Animals↗
PubMed2004

Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic beta-cell ATP-sensitive K+ currents.

Hypoglycemic sulfonylureas (e.g. tolbutamide, glibenclamide) exert their stimulatory effects on pancreatic beta-cells by closure of ATP-sensitive K(+) (K(ATP)) channels. Pancreatic K(ATP) channels are composed of two subunits, a pore-forming inwardly rectifying K(+) channel (Kir6.2) subunit and a regulatory subunit (the sulfonylurea receptor of subtype 1 (SUR1)) in a (SUR1/Kir6.2)(4) stoichiometry. The aim of the present study was to characterize the interaction of green-fluorescent 3-[3-(4,4 difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacen-3-yl)propanamido] glibenclamide (Bodipy-glibenclamide) with pancreatic beta-cell K(ATP) channels using patch-clamp and fluorescence microscopy techniques. Bodipy-glibenclamide inhibited K(ATP) currents from the clonal insulinoma cell line RINm5F half-maximally at a concentration of 0.6nM. Using laser-scanning confocal microscopy Bodipy-glibenclamide was shown to induce a diffuse fluorescence across the RINm5F cell, but only about 17% of total Bodipy-glibenclamide-induced fluorescence intensity in RINm5F cells was due to specific binding to SUR1. Using fluorescence correlation spectroscopy, it could be demonstrated that the fluorescence label contributes to the protein binding and, therefore, possibly also to the non-specific binding of Bodipy-glibenclamide observed in RINm5F cells. Specific binding of Bodipy-glibenclamide to SUR1 in RINm5F cells might be localized to different intracellular structures (nuclear envelope, endoplasmic reticulum, Golgi compartment, insulin secretory granules) as well as to the plasma membrane. In conclusion, Bodipy-glibenclamide is a high-affinity blocker of pancreatic beta-cell K(ATP) currents and can be used for visualizing SUR1 in intact pancreatic beta-cells, although non-specific binding must be taken into account in confocal microscopy experiments on intact beta-cells.

Animals↗
PubMed2006

A longitudinal case series investigating cellular changes to the transplanted cornea using confocal microscopy.

PURPOSE: To perform a longitudinal evaluation of subjects who had undergone penetrating keratoplasty, using slit scanning confocal microscopy. METHODS: In vivo confocal microscopy was used to evaluate the central cornea of four subjects who had recently undergone penetrating keratoplasty. Subjects were examined on four occasions over a 12-month period after surgery. Quantitative and qualitative aspects of corneal morphology were compared against data from normal control subjects. RESULTS: The epithelium varied in appearance between subjects and took at least 12 months to return to a similar arrangement to that seen in normal eyes. Bowman's layer was viewed as an acellular layer immediately after surgery with no evidence of nerve fibres, although nerve components were apparent 12 months after surgery. Stromal nerves were not visible immediately after surgery. One year following penetrating keratoplasty there was evidence of thin nerves running a straight course through the central stroma. Keratocyte density in the anterior and posterior stroma was lower in the transplanted cornea but appeared to remain constant over a period of 1 year. Activated keratocytes were seen in the anterior stroma of all subjects; they appeared to be responsible for significant levels of corneal haze. The time period within which this keratocyte activation occurred varied between individuals. Endothelial cell density decreased at an accelerated rate over the 12-month period. CONCLUSIONS: Confocal microscopy allows cellular changes to be monitored in vivo following penetrating keratoplasty and may assist clinicians in understanding postoperative recovery.

Adult↗
PubMed2006

Study of the cholesterol-GM3 ganglioside interaction by surface pressure measurements and fluorescence microscopy.

The nature of the cholesterol/glycolipid interaction in rafts being poorly understood, the interaction of cholesterol with the GM(3) ganglioside has been studied by surface pressure measurements and fluorescence microscopy. Results have been compared to those obtained with sphingomyelin (SM)-cholesterol and palmitoyl-oleoyl-phosphatidylcholine (POPC)-cholesterol monolayers. The analysis of (pi-A) isotherms of mixed monolayers show a condensing effect of cholesterol on GM(3) molecules, in the same range than the effect observed with POPC and higher than the effect on SM. This is likely due to the similar state of GM(3) and POPC, since both molecules are in liquid expanded phases in our experimental conditions. The study of the cholesterol desorption induced by beta-cyclodextrin suggests also that the GM(3)-cholesterol interaction is rather weak as in the case of POPC-cholesterol interaction, and clearly lower than SM-cholesterol one. This lack of interaction is discussed in terms of nature of lipid chains and molecular shape, and suggests that no hydrogen bond is formed between GM(3) and cholesterol polar heads. Fluorescence microscopy performed on mixed GM(3)-cholesterol monolayers shows the presence, at surface pressure higher than 10 mN/m, of particular blurring patterns without defined boundary, which could be due to a partial solubilization in one phase of different phases observed at lower surface pressure, whereas SM-cholesterol and POPC-cholesterol monolayers are homogeneous at the lateral resolution of our microscopy set-up.

Cholesterol↗
PubMed2004

Novel techniques in electron microscopy.

Emerging techniques in electron microscopy promise to yield a wide range of new information about the nervous system. Aided by the development of detectors, electron optics, energy filters, computer automation and preparative methods, electron tomography now provides three-dimensional structures over a scale ranging from single receptor molecules to synapses and neurons. To relate structure to function, a variety of correlative methods are being developed, including protein tags observable both by light microscopy of living cells and, subsequently, by electron microscopy. It is also becoming possible to measure functionally important ions like Ca(2+) in cellular compartments at a scale of about 10 nm by exploiting new advances in electron energy loss and X-ray spectroscopic imaging.

Animals↗
PubMed2006

Visualizing the conversion of carbamazepine in aqueous suspension with and without the presence of excipients: a single crystal study using SEM and Raman microscopy.

Visual observations of the hydration process of single carbamazepine (CBZ) crystals in water and in various excipient solutions [(1% w/v) - hydroxypropyl cellulose (HPC), poly(vinyl pyrrolidone) (PVP), sodium carboxymethylcellulose (CMC) at pH 7.5 and 3.0, and polyethylene glycol (PEG)] using scanning electron microscopy (SEM) are reported in this paper. Raman microscopy was used to confirm the chemical structures of the unconverted CBZ and the CBZ dihydrate (DH) needles. It was found that defect structures were a more important driving force than the nature of crystal faces for the initiation of the hydration, but face differences became obvious after 6 h immersion. The biggest crystal face grown from methanol, (100), was the slowest one to be covered with DH needles. A comparison of the molecular arrangements along the three crystal faces [(100), (010) and (001)] was carried out using crystal structure visualization software, and fewer polar groups exposed on the (100) face than on the (001) and (010) faces were found, explaining the comparatively weak interaction of the (100) face with water during hydration. Furthermore, investigation of the influence of excipients on the hydration of CBZ showed that both HPC and PVP strongly inhibited conversion, and no conversion of CBZ to DH was found after 18 h immersion in water. PEG and CMC (pH 7.5) were less potent inhibitors than HPC and PVP, and DH needles were observed on all the faces except the (100) face after 18 h immersion. No conversion was detected for the crystal immersed in CMC solution at pH 3.0. This is likely to be caused by the decreased polarity of CMC in water at pH 3.0 (pKa,cmc = 4.3), and thus a higher surface adsorption of CMC to the CBZ crystals in dispersion. The influence of excipients on the conversion of CBZ observed in this study agreed well with our previous quantitative studies using Raman spectroscopy. In this study, visual observation using electron microscopy has been demonstrated to be a unique and powerful tool to improve our understanding of polymorphic conversions of CBZ in aqueous suspension.

Carbamazepine↗
PubMed2005

Clinical and microstructural analysis of patients with hyper-reflective corneal endothelial nuclei imaged by in vivo confocal microscopy.

The purpose of this study was to determine the significance of hyper-reflective corneal endothelial nuclei imaged by in vivo confocal microscopy. A retrospective analysis was performed using a database of 505 patients that had undergone in vivo confocal microscopy of the cornea. All subjects with hyper-reflective endothelial nuclei were identified and these images were analysed to determine corneal endothelial cell density and morphology. The clinical notes of these patients were reviewed and corresponding data regarding corneal thickness was obtained from a related database of Orbscan II pachymetry. Hyper-reflective endothelial nuclei were identified in 41 eyes of 39 (7.7%) patients. Diagnoses included previous cataract surgery or penetrating keratoplasty, posterior polymorphous dystrophy, Fuchs' endothelial dystrophy and irido-corneal endothelial syndrome. No patients with clinically normal corneas exhibited bright endothelial nuclei. The mean endothelial cell density in this group was 1325+/-872 cells mm(-2) and endothelial density was below age-adjusted normal values in 69.2% of patients. Both cellular polymegathism (coefficient of variation of cell area 33.9+/-7.4%) and cellular pleomorphism were noted (51.8+/-9.0% hexagonal cells). The mean central corneal thickness was 582+/-52 microm. There was no significant difference in endothelial density and morphology compared to cases that had low endothelial density but did not exhibit bright nuclei. In conclusion, this study is the first to investigate the significance of bright endothelial nuclei detected by in vivo confocal microscopy. The strong association with corneal disease states suggests that the most likely explanation for this appearance is the alteration in cellular/nuclear morphology, composition or function.

Cataract Extraction↗
PubMed2005

Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis.

Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (-) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey.

Adolescent↗
PubMed2005

Fourier transform infrared microscopy and imaging: detection of fungi in wood.

FTIR microscopy was used to detect and discriminate the two wood decaying fungi Trametes versicolor and Schizophyllum commune in experimentally infected beech wood blocks. The distribution of fungal mycelium in wood was locally resolved and semiquantitatively recorded using FTIR microscopy combined with a focal plane array detector and image analysis. Cluster analysis revealed major differences between FTIR spectra recorded from wood fibers and empty vessel lumina and spectra from mycelium of both fungal species, irrespective of whether the fungi were grown on the surface of wood or inside vessel lumina. Species-specific clustering of spectra of fungal mycelium grown on the wood surface and inside vessel lumina demonstrated the potential of FTIR microscopy to discriminate among fungal species decaying wood.

Cluster Analysis↗
PubMed2005

Reflectance-mode confocal microscopy of pigmented skin lesions--improvement in melanoma diagnostic specificity.

BACKGROUND: In vivo confocal microscopy enables skin visualization with a quasihistopathologic resolution. OBJECTIVE: We sought to describe confocal features in melanocytic lesions and to evaluate their diagnostic significance for melanoma (MM) identification. METHODS: Thirty seven MMs, 49 acquired nevi, and 16 Spitz/Reed nevi, presenting equivocal clinicodermoscopic aspects were investigated by confocal microscopy. RESULTS: MMs and nevi significantly differed for some aspects. In multivariate analysis, the presence of nonedged dermal papillae, atypical cells, and isolated nucleated cells within dermal papilla, pagetoid cells, widespread pagetoid infiltration, and cerebriform clusters were strongly correlated with MM diagnosis. A receiver operating characteristic curve value of 0.952 was obtained. LIMITATIONS: Spitz/Reed nevi represented a pitfall in confocal diagnosis, owing to the frequent observation of pagetoid infiltration, architectural disarray, and cytologic atypia, and to the impossibility of evaluating cell maturation with depth. CONCLUSION: Characterization of confocal microscopy features of MMs and nevi seems to improve diagnostic accuracy for melanocytic lesions that are difficult to diagnose.

Diagnosis, Differential↗
PubMed2004

Quality assessment model of 3 different microkeratomes through confocal microscopy.

PURPOSE: To study the quality of the cut created by 3 microkeratomes from 2 different generations using corneal confocal microscopy. SETTING: Department of Refractive Surgery, Instituto Oftalmológico de Alicante, Alicante, Spain. METHODS: Two different studies were conducted: Study 1 and Study 2. Study 1 was a prospective analysis using confocal microscopy examination data from 2 reference groups: Group A (control) with 20 nonoperated eyes of 20 healthy volunteers, and Group B with 50 eyes of 30 patients operated on with the Bausch & Lomb Automated Corneal Shaper (ACS) microkeratome. Study 2 was a prospective randomized double-masked study in which 40 eyes of 20 patients underwent myopic laser in situ keratomileusis by 1 surgeon. The right and left eyes of each patient were randomly and alternatively assigned to Group C, flap made with the Hansatome microkeratome, or Group D, flap made with the Moria M2. Immediately after surgery, the microscopic appearance of the cut was subjectively evaluated by the surgeon. One month postoperatively, flap thickness, particle density, and the subclinical confocal wound healing opacity (WHO) index were evaluated with the corneal confocal microscope. RESULTS: Surgeon handling comfort was nearly the same with the Hansatome as with the M2 (P =.540). However, the apparent quality of the resulting flap was better with the M2 microkeratome (P =.041). The depth of the cuts made by the 3 microkeratomes were significantly different (P<.001), with the ACS flaps being thinner than the flaps made with the Hansatome or the M2. Particle density at the interface was significantly poorer in the eyes operated on with the ACS, but in these cases the WHO index was significantly greater (P<.001 in both cases). CONCLUSIONS: Confocal microscopy is a very useful tool to evaluate the quality of the cut made by different microkeratomes. Overall, the predictability in flap thickness and the apparent quality of the cut made by the Moria M2 microkeratome are better than those obtained with the Hansatome or the ACS. The thin flap made by the ACS microkeratome produced a significantly greater WHO index than the thicker flaps created with the other 2 microkeratomes.

Corneal Stroma↗
PubMed2005

Classification of single-projection reconstructions for cryo-electron microscopy data of icosahedral viruses.

We present a novel strategy for classification of heterogeneous electron microscopy data of icosahedral virus particles. The effectiveness of the procedure, which is based on classification of single-projection reconstructions (SPRs), is first investigated using simulated data. Of several reconstruction approaches examined, best results were obtained with algebraic reconstruction techniques (ART) when providing prior information about the reconstruction in the form of a starting volume. The results presented indicate that SPR-classification is sufficiently sensitive to classify assemblies with differences of only a few percent of the total mass. The usefulness of this procedure is illustrated by application to a heterogeneous cryo-electron microscopy dataset of adenovirus mutant dl313, lacking minor coat protein IX. These data were successfully divided into two distinct classes, in agreement with gel analysis and immuno-electron microscopy results. The classes yielded a wildtype-like reconstruction and a reconstruction representing the polypeptide IX-deficient dl313 virion. As the largest difference between these volumes is found at the location previously assigned to the external portion of minor coat protein polypeptide IIIa, questions arise concerning the current adenovirus model.

Adenoviruses, Human↗
PubMed2006

Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles.

Synchrotron-based X-ray fluorescence microscopy (microXRF) is a powerful tool to study the two-dimensional distribution of a wide range of biologically relevant elements in tissues and cells. By growing mouse fibroblast cells directly on formvar-carbon coated electron microscopy grids, microXRF elemental maps with well-defined subcellular resolution were obtained. In order to colocalize the elemental distribution with the location of specific cellular structures and organelles, we explored the application of a commercially available secondary antibody conjugated to FluoroNanogold, a dual-label that combines a regular organic fluorophore with a 1.4 nm Au-cluster as xenobiotic label for microXRF imaging. Adherent mouse fibroblast cells were grown on silicon nitride windows serving as biocompatible XRF support substrate, and labeled with FluoroNanogold in combination with primary antibodies specific for mitochondria or the Golgi apparatus, respectively. Raster scanning of the in-air dried cells with an incident X-ray energy of 11.95 keV, sufficient to ensure excitation of the Au Lalpha line, provided two-dimensional maps with submicron resolution for Au as well as for most biologically relevant elements. MicroXRF proved to be sufficiently sensitive to image the location and structural details of the Au-labeled organelles, which correlated well with the subcellular distribution visualized by means of optical fluorescence microscopy.

Animals↗
PubMed2006

Macromolecular mass spectrometry and electron microscopy as complementary tools for investigation of the heterogeneity of bacteriophage portal assemblies.

The success of electron-cryo microscopy (cryo-EM) and image reconstruction of cyclic oligomers, such as the viral and bacteriophage portals, depends on the accurate knowledge of their order of symmetry. A number of statistical methods of image analysis address this problem, but often do not provide unambiguous results. Direct measurement of the oligomeric state of multisubunit protein assemblies is difficult when the number of subunits is large and one subunit renders only a small increment to the full size of the oligomer. Moreover, when mixtures of different stochiometries are present techniques such as analytical centrifugation or size-exclusion chromatography are also less helpful. Here, we use electrospray ionization mass spectrometry to directly determine the oligomeric states of the in vitro assembled portal oligomers of the phages P22, Phi-29 and SPP1, which range in mass from 430 kDa to about 1 million Da. Our data unambiguously reveal that the oligomeric states of Phi-29 and SPP1 portals were 12 and 13, respectively, in good agreement with crystallographic and electron microscopy data. However, in vitro assembled P22 portals were a mixture of 11- and 12-mer species in an approximate ratio of 2:1, respectively. A subsequent reference-free alignment of electron microscopy images of the P22 portal confirmed this mixture of oligomeric states. We conclude that macromolecular mass spectrometry is a valuable tool in structural biology that can aide in the determination of oligomeric states and symmetry of assemblies, providing a good starting point for improved image analysis of cryo-EM data.

Bacteriophages↗
PubMed2005

Use of light, scanning electron microscopy and bioassays to evaluate parasitism by entomopathogenic fungi of the red scale insect of palms (Phoenicococcus marlatti Ckll., 1899).

We have evaluated the parasitism of the red scale insect of the date palm (Phoenicococcus marlatti) by entomopathogenic fungi, using light microscopy (LM), scanning electron microscopy (SEM) and low temperature scanning electron microscopy (LTSEM). Beauveria bassiana, Lecanicillium dimorphum and Lecanicillium cf. psalliotae, were inoculated directly on the scale insects or on insect infested plant material. We found that L. dimorphum and L. cf. psalliotae developed on plant material and on scale insects, making infection structures. B. bassiana was a bad colonizer of date palm leaves (Phoenix dactylifera L.) and did not parasite the scale insects.

Cocos↗
PubMed2005

Scanning probe microscopy examination of the surface properties of keratin fibres.

A Scanning Probe Microscope (SPM) was used to investigate the mechanical properties of the surface of hair and wool fibres. Using stiff cantilevers, penetration was achieved on the fibres under ambient conditions and at increased relative humidity (RH). The Young's modulus of the exocuticle was estimated to be 2.1GPa under ambient conditions, decreasing to 0.6GPa at 96% RH. Contrary to findings by other authors [Crossley, J.A.A., Gibson, C.T., Mapledoram, L.D. , Huson, M.G., Myhra, S., Pham, D.K., Sofield, C.J., Turner, P.S., Watson, G.S., 2000. Atomic force microscopy analysis of wool fibre surfaces in air and under water. Micron 31, 659-667; Gibson, C.T., Watson, G.S., Mapledoram, L.D., Kondo, H., Myhra, S., 1999. Characterisation of organic thin films by atomic force microscopy-application of force vs. distance analysis and other modes. Applied Surface Science 144-145, 618-622; Blach, J., Loughlin, W., Watson, G.S., Myhra, S., 2001. Surface characterization of human hair by atomic force microscopy in the imaging and f-d modes. Journal of Cosmetic Science 23, 165-174], the surface lipid layer could not be penetrated using soft cantilevers in force-distance (f-d) mode in water. Attempts were made to remove the lipid layer from the surface both physically and chemically so as to examine the influence of the lipid on f-d measurements. Using both techniques, it was not possible to remove lipid without damaging the fibre, suggesting that the lipid is an integral part of the surface rather than a discrete surface layer. Adhesion measurements on the surface of wool, nylon and polyethylene, showed that in water and at high RH, the surface of keratin fibres is more akin to a polyamide. At low RH and in liquid paraffin, the surface is more akin to a hydrocarbon, suggesting it is capable of altering its structure in response to different environments.

Animals↗
PubMed2004

Low-voltage electron microscopy of polymer and organic molecular thin films.

We have demonstrated the capabilities of a novel low-voltage electron microscope (LVEM) for imaging polymer and organic molecular thin films. The LVEM can operate in transmission electron microscopy, scanning transmission electron microscopy, scanning electron microscopy, and electron diffraction modes. The microscope operates at a nominal accelerating voltage of 5 kV and fits on a tabletop. A detailed discussion of the electron-sample interaction processes is presented, and the mean free path for total electron scattering was calculated to be 15 nm for organic samples at 5 kV. The total end point dose for the destruction of crystallinity at 5 kV was estimated at 5 x 10(-4) and 3.5 x 10(-2) C/cm2 for polyethylene and pentacene, respectively. These values are significantly lower than those measured at voltages greater than 100 kV. A defocus series of colloidal gold particles allowed us to estimate the experimental contrast transfer function of the microscope. Images taken of several organic materials have shown high contrast for low atomic number elements and a resolution of 2.5 nm. The materials studied here include thin films of the organic semiconductor pentacene, triblock copolymer films, single-molecule dendrimers, electrospun polymer fibers and gold nanoparticles.

Electrons↗
PubMed2004

Identification of hibernating myocardium by acoustic microscopy.

Hibernating myocardium is viable myocardium that recovers after revascularization. The observation of loss of contractile proteins (myofibrils) and accumulation of glycogen in hibernating cardiomyocytes provide the basis for diagnosing hibernating myocardium. In this pilot study, acoustic microscopy was used to identify the cellular structure of normal vs. hibernating myocardium. Sections cut at 5-microm of archival paraffin blocks on glass slides were used for this study. Acoustic microscopy of normal cardiomyocytes showed intracellular linear echoes suggestive of myofibrils, and cardiomyocytes of hibernating myocardium revealed absence of myofibrils and dense intracellular echoes that corresponded to glycogen accumulation on optical microscopy. This modality of visualization allows a definitive diagnosis of hibernating myocardium.

Glycogen↗